the terms "antigenic determinant" and "epitope" are defined as the parts of a molecule that can interact specifically with either the cellular or the humoral products associated with the immune response. The term "antigen" is defined as anything that can serve as a target for an immune response. The immune response can be either cellular or humoral. The term "cell mediated immunity" is defined as an immune response mediated by cells rather than by antibody. The term "delayed type hypersensitivity" is defined as a T-lymphocyte-mediated inflammatory response that occurs in close proximity to the site of injection or application of the inciting antigen. It includes, but is not limited to, delayed type hypersensitivity and cytotoxic T cells. A "hapten" is defined herein as a substance that reacts selectively with appropriate antibodies or T cells but the hapten itself is usually not immunogenic. Most haptens are small molecules or small parts of large molecules, but some macromolecules can also function as haptens. The term "antibody" means molecules that also bind antigens, however, they are distinguished from transfer factor in that the molecular weight of antibodies is between approximately 160,000 Daltons and 1,000,000 Daltons.
Transfer factors have been defined as a dialyzable material or family of materials that can be extracted from lymphoid cells of humans and certain other animals and have the capacity to transfer immune responses from one individual to another, even across species. The material is a substance obtained from leukocytes, usually lysed, from humans and other vertebrates that have been sensitized so as to express delayed type hypersensitivity or other cell mediated responses to a sensitizing antigen. Transfer factor binds homologous antigen and has the capacity to mediate transfer of delayed type hypersensitivity and other cell mediated immune responses from one individual to another. In such a situation the individual from whom the transfer factor is obtained has been sensitized to the antigen of interest.
Notwithstanding the above properties, the transfer factors are smaller than antibodies, and do not transfer antibody mediated responses, nor do they induce antibody production..sup.1,2,3 These properties of transfer factor are also described by Spiller et al. which discusses a "transfer factor" secured from the leukocytes of healthy donors..sup.4 The material suppresses disease symptoms. Spiller et al. describe the material as being heat stable, and having a molecular weight of less than 20,000 Daltons. It is secured by lysing leukocytes, and then incubating the lysate with Mg.sup.+2 and DNase, followed by filtration through a millipore filter. FNT .sup.1 Kirkpatrick et al., J. Immunol. 134:1723-1727 11985) FNT .sup.2 Peterson et al., J. Immunol. 126:2480-2484 (1981) FNT .sup.3 Burger et al., Cell Immunol. 29:410-413 (1977) FNT .sup.4 U.S. Pat. No. 3,991,182 to Spiller, et at.
There have been numerous additional attempts to characterize the substance referred to as transfer factor, these being reported in both the scientific and patent literature. In all of these reports, the transfer factor material was a crude fraction of cell lysate. To the inventors knowledge, no one has produced a substantially pure transfer factor. Baram et al. fractionated human leukocyte extracts through ion exchange chromatography, using diethylaminoethyl cellulose (DEAE)..sup.5 This work was continued and, as reported by Baram et al., gel filtration and paper chromatography were used to further fractionate human leukocyte extracts..sup.6 Among the conclusions presented by this work was that transfer factor contained nucleosides. Work by Lawrence et al. using gel filtration chromatography on leukocyte extracts of sensitized humans, led to a proposal that transfer factor is (i) water soluble, (ii) dialyzable, (iii) has a molecular weight of less than 10,000 Daltons, (iv) was resistant to deoxyribonuclease, ribonuclease and trypsin digestion, and (v) possessed a chromatographic peak showing greater absorbance at 260 nm than at 280 nm..sup.7,8,9 This combination of factors led to a proposal that transfer factor was a small, ribonuclease resistant polyribonucleotide. FNT .sup.5 Baram et al., J. Allergy 33:498-506 (1962) FNT .sup.6 Baram et al., J. Immunol. 97:407-420 (1966) FNT .sup.7 Lawrence et al., J. Clin. Invest. 34:219-232 (1955); FNT .sup.8 Lawrence et al., J. Exp. Med. 104:321321 (1956) FNT .sup.9 Lawrence et al., Trans. Assoc. Amer. Physicians 76:84-89 (1963)
Progress toward the molecular characterization of transfer factors has been slow, limited largely by the lack of an adequate purification methodology and the need for quantitative assay methods. It has been shown that molecules having transfer factor activity are relatively small, i.e. less than 6000 Daltons, hydrophilic, and polar in native form. Furthermore, transfer factor activity survives heating at 56.degree. C., but not at 75.degree. C., for 30 minutes, and at least brief exposure to 95% ethanol. Results from enzyme sensitivity and activity depletion studies have produced results compatible with a nucleopeptide or nucleoprotein model for the structure of transfer factors. Caution must be used in interpreting prior art results, however, because impure preparations were studied and quantitative determinations were not performed. Thus, the molecular nature of transfer factors is, as yet, not well understood.
The inclination toward the assumption that a nucleotide or nucleoside was a part of the transfer factor molecule was continued by Gottlieb et al..sup.10,11,12 Gottlieb differentiated transfer factors from immune modulators (the '379 patent), and amplifiers (the '079 patent). In the Lancet publication, Gottlieb postulated that transfer factor consisted of 12 amino acids and an oligonucleotide. As a result, research focused on the study of eluates at wavelengths of 254 nm or greater. Many reports noted high 254/260 nm to 280 nm absorbance ratios, again suggesting oligonucleotides as part of the transfer factor fraction..sup.13,14,15,16,17,18,19,20,21 Similarly, Warren hypothesizes a molecule of a molecular weight of from 5000 to 10,000 Daltons containing protein and RNA..sup.22 Goust et al. describes dialyzable transfer factor as a mixture of molecules of molecular weight generally from 4000 to 7000 Daltons and containing a ribonucleotide..sup.23 Again, Wilson et al..sup.24 describes three forms of transfer factor, all of which contain a nucleotide moiety and a peptide moiety. (Note column 11 of this reference.) FNT .sup.10 Gottlieb et al., Lancet 2:822-813 (1973) FNT .sup.11 U.S. Pat. No. 4,468,379 to Gottlieb, et al. FNT .sup.12 U.S. Pat. No. 4,616,079 to Gottlieb, et al. FNT .sup.13 Arala-Chaves et al., Int. Arch. Allergy 31:353-365 (1967) FNT .sup.14 Neidhart et al., Cell Immunol. 9:319-323 (1973) FNT .sup.15 Reymond et al., Vex sang. 29:338-351 (1975) FNT .sup.16 Dunnick et al., Proc. Natl. Acad. Sci. USA 72:4573-4576(1975) FNT .sup.17 Vandenbark et al., J. Immunol. 118:636-641 (1977) FNT .sup.18 Dunnick et al., J. Immunol. 118:1944-1950 (1977) FNT .sup.19 Burger et at., J. Immunol. 122:1091-1098 (1979) FNT .sup.20 Wilson, Trans. Assoc. Amer. Physicians 92:239-256 (1979) FNT .sup.21 Borvak et at., Acta Virol. 29:119-128 (1985) FNT .sup.22 U.S. Pat. No. 4,435,384 to Warren FNT .sup.23 U.S. Pat. No. 4,001,080 to Goust, et al. FNT .sup.24 U.S. Pat. No. 4,816,563 to Wilson, et al.
The progress that has been made in characterizing the impure transfer factor material is summarized in a review by Kirkpatrick..sup.25 In that review, the dialyzable material that contains transfer factor activity is described as a polypeptide with a molecular weight of between 4000 and 6000 Daltons and is protease sensitive. The transfer factor material apparently binds specifically to antigen..sup.26 The review states that the presence of nucleic acids, ribose, and phosphodiester groups has not been ruled out..sup.27 Thus, the physical characteristics of the transfer factor material could not be definitively determined because substantially pure material had not been isolated. FNT .sup.25 Kirkpatrick, J. Allergy Clin Immunol, 81:803-813, 1988 FNT .sup.26 Borkowsky,et al., J. Immunol. J. 26:486-489, 1981. FNT .sup.27 Kirkpatrick, "Transfer Factor" Supra. at page 808
It will be seen that the prior art in this field has suggested that transfer factor is a nucleotide/protein complex. However, because a substantially pure transfer factor has not been isolated, it is not possible to conclusively characterize the transfer factor material.
Interest in the molecule and its structure has, if anything, increased because of its therapeutic efficacy. Apart from therapeutic uses described by the references set forth above, reference may be made, e.g., to Viza et al..sup.28 suggesting transfer factor therapy for Herpes simplex virus. One also notes Warren.sup.29, describing dermatological efficacy for blemishes, ache, condyloma and HSV. The transfer factor fraction has been shown to be efficacious against C. albicans, as shown in Kirkpatrick et al,.sup.30 the disclosure of which is incorporated by reference. Additional showings of efficacy against Herpes simplex may be found..sup.31,32,33 Varicella zoster infection has been prevented with transfer factor..sup.34 Transfer factor has showed efficacy against cryptosporidiosis in AIDs patients..sup.35,36 All of these studies were performed with only partially purified transfer factor fractions. No clinical or biochemical studies have been performed to date with substantially pure transfer factor material because of the difficulty in isolating and characterizing pure transfer factor material. FNT .sup.28 European Patent Application 101,200 to Viza et al. FNT .sup.29 U.S. Pat. No. 4,435,384 FNT .sup.30 Kirkpatrick et al., in Khan et al., ed., Immune Regulators In Transfer Factor, pg. 547-559 (Academic Press, 1979) FNT .sup.31 Khan et al., Dermatologica 163:177-185 (1981) FNT .sup.32 Dwyer in Kirkpatrick et al., Immunobiology of Transfer Factor, pg. 233-243 (Academic Press, 1983) FNT .sup.33 Viza et al., Lymphokine Res 4:27-30 (1985) FNT .sup.34 Steele et al, New Eng. J. Med. 303:355-359 (1980) FNT .sup.35 Louie, et al., Clin, Immunol. lmmunopath 44:329-334 (1987) FNT .sup.36 McMeeking et al., J. Infect. Dis 161:108-112 (1990)
As can be seen by the foregoing review of the literature on transfer factor, the isolation and characterization of a substantially pure transfer factor material has eluded the research community for over thirty years. Despite keen scientific and clinical interests, and after deducing several important physical parameters about .the elusive transfer factor material, the actual physical isolation of substantially pure material has not been possible.
What is needed is substantially pure transfer factor material. With substantially pure material in hand, it would be possible to sequence the material and produce the material either chemically or by recombinant methods. These molecules could then be administered to humans or animals thereby transferring immunity to a specific antigen or epitope. Substantially pure transfer factor could be made to treat already infected humans or animals or could be used to prevent disease. Isolation and purification of substantially pure transfer factor would be of great benefit to the treatment of disease.